Frequently asked questions (FAQ) for Illumina DNA Prep with Exome 2.5 Enrichment v2

What improvements are there to the Illumina DNA Prep with Exome 2.5 Enrichment v2 workflow?

• Illumina DNA Prep with Exome 2.5 Enrichment v2 has improved uniformity, reduced GC dropout, and a lower fold-80 base penalty (fold over-coverage necessary to raise 80% of bases in non-zero coverage targets to the mean coverage level in those targets) compared to the v1 kit.

• These improvements are achieved by updated amplification reagents.

Specifically, which product contents and workflow changes achieve these differences?

• The V2 kit contains LAM (Library Amplification Mix) and DDR (DNA Denaturing Reagent) in place of EPM (Enhanced PCR Mix), used in both the "Amplify Tagmented DNA" and "Amplify Enriched Library" sections of the protocol. LAM and DDR are combined together in a master mix before use. Note: LAM is expected to have a mild odor, a cloudy appearance, and high viscosity.

• In V2, TWB2 is used in place of TWB.

• The V2 kit utilizes SMB4 beads, and bead volume is reduced from 250 µl (v1 protocol) to 200 µl (v2 protocol) in the capture step, with a note that users may reduce the volume to a minimum of 100 µl. This volume reduction is expected to improve coverage of high-GC targets, but may impact SNV and indel recall rates. As this can vary with sample type and loci, labs will need to optimize when using volumes less than 200 µl. Additional details and data are provided in the Illumina Knowledge article Workflow Optimization by Modulating SMB4 Volume for Illumina DNA Prep with Exome 2.5 Enrichment v2.

Since a lower volume of SMB4 is used, is the same amount still supplied in the kit?

• Yes, the same amount of SMB4 is still supplied compared to the v1 kit.

What are the critical steps in the workflow?

• Handling of LAM reagent. Due to high viscosity, it is important to mix the LAM reagent well before use. Thorough, gentle pipette mixing is recommended to avoid excessive bubbles.

• Mixing well the LAM enzyme + DDR before use. Thorough vortexing of combined LAM+DDR is recommended. Lower than expected library yield and low coverage and enrichment metrics can result from insufficient mixing.

• SMB4 volume.200 µl is the default and the strongly recommended volume to use. Though if labs see lower than desired coverage of critical high GC regions, this volume may be reduced to as low as 100 µl to try to improve coverage of these regions. This volume reduction is expected to improve coverage of high-GC targets, but may impact SNV and indel recall rates. As this can vary with sample type and loci, labs will need to optimize when using volumes less than 200 µl. Additional details and data are provided in the Illumina Knowledge article Workflow Optimization by Modulating SMB4 Volume for Illumina DNA Prep with Exome 2.5 Enrichment v2.

Are data available (demo data, datasheet, etc.) that shows concordance of v2 to v1 performances?

• The datasheet contains comparison data for v1 and v2 kits on coverage uniformity, GC dropout and Fold-80 penalty (Datasheet Figure 6).

• BaseSpace Demo Data will also contain datasets for the v1 (on-market) kit as well as the new v2 kit.

How many samples are recommended per sequencing run?

• This information can also be found in the datasheet.

Are there any updates to the analysis pipeline required for the v2 kit?

• No, there are no updates to the software/analysis pipeline, nor the manifest file.

Is there an Illumina-provided or default Panel of Normals (PON) for CNV calling?

• There is no PON supplied by Illumina for CNV calling. Customers can utilize an in-run PON created from samples from the same sequencing run and library prep as the case samples, provided there are at least 30 samples. For details, see DRAGEN 4.4 documentation.

For any feedback or questions regarding this article (Illumina Knowledge Article #10119), contact Illumina Technical Support [email protected].